Many nucleic acid stains show a strong fluorescence enhancement upon binding to double-stranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of ∼14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed
Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures.